Nucleic acids encoding active and inactive CCR5 chemokine receptors

ABSTRACT

A peptide has an amino acid sequence having more than 80% homology with the amino acid sequence listed as SEQ ID NO:4. A nucleic acid molecule has more than 80% homology with one of the nucleic acid sequences listed as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. Ligands, anti-ligands, cells vectors relating to the peptide and/or nucleic acid molecule are also used.

This application is a division and claims priority under 35 U.S.C. §120 to U.S. patent application Ser. No. 09/626,939, filed Jul. 27, 2000, which is a continuation and claims priority under 35 U.S.C. §120 to U.S. patent application Ser. No. 08/833,752, filed Apr. 9, 1997, now U.S. Pat. No. 6,448,395, which claims priority under 35 U.S.C. §119(a)-(d) to EP 96870021.1, filed Mar. 1, 1996, and EP 96870102.9, filed Aug. 6, 1996.

BACKGROUND

1. Field of the Invention

The present invention concerns new peptides and the nucleic acid molecules encoding said peptides, the vector comprising said nucleic acid molecules, the cells transformed by said vector, inhibitors directed against said peptides or said nucleic acid molecules, a pharmaceutical composition and a diagnostic and/or dosage device comprising said products, and non human transgenic animals expressing the peptides according to the invention or the nucleic acid molecules encoding said peptides.

The invention further provides a method for determining ligand binding, detecting expression, screening for drugs binding specifically to said peptides and treatments involving the peptides or the nucleic acid molecules according to the invention.

2. Technological Background of the Art

Chemotactic cytokines, or chemokines, are small signalling proteins that can be divided in two subfamilies (CC- and CXC-chemokines) depending on the relative position of the first two conserved cysteines. Interleukin 8 (IL-8) is the most studied of these proteins, but a large number of chemokines (Regulated on Activation Normal T-cell Expressed and Secreted (RANTES), Monocyte Chemoattractant Protein 1 (MCP-1), Monocyte Chemoattractant Protein 2 (MCP-2), Monocyte Chemoattractant Protein 3 (MCP-3), Growth-Related gene product a (GROα), Growth-Related gene product β (GRO)β, Growth-Related gene product γ (GROγ), Macrophage Inflammatory Protein 1α (MIP-1α) and β, etc.) has now been described [4]. Chemokines play fundamental roles in the physiology of acute and chronic inflammatory processes as well as in the pathological dysregulations of these processes, by attracting and simulating specific subsets of leucocytes [32]. RANTES for example is a chemoattractant for monocytes, memory T-cells and eosinophils, and induces the release of histamine by basophils. MCP-1, released by smooth muscle cells in arteriosclerotic lesions, is considered as the factor (or one of the factors) responsible for macrophage attraction and, therefore, for the progressive aggravation of the lesions [4].

MIP-1α, MIP-1β and RANTES chemokines have recently been described as major HIV-suppressive factors produced by CD8⁺ T-cells [9]. CC-chemokines are also involved in the regulation of human myeloid progenetor cell proliferation [6, 7].

Recent studies have demonstrated that the actions of CC- and CXC-chemokines are mediated by subfamilies of G protein-coupled receptors. To date, despite the numerous functions attributed to chemokines and the increasing number of biologically active ligands, only six functional receptors have been identified in human. Two receptors for interleukin-8 (IL-8) have been described [20, 29]. One (IL-8RA) binds IL-8 specifically, while the other (IL-8RB) binds IL-8 and other CXC-chemokines, like GRO. Among receptors binding CC-chemokines, a receptor, designated CC-chemokine receptor 1 (CCR1), binds both RANTES and MIP-1α [31], and the CC-chemokine receptor 2 (CCR2) binds MCP-1 and MCP-3 [8, 44, 15]. Two additional CC-chemokine receptors were cloned recently: the CC-chemokine receptor 3 (CCR3) was found to be activated by RANTES, MIP-1α and MIP-1β [10]; the CC-chemokine receptor 4 (CCR4) responds to MIP-1, RANTES and MCP-1 [37]. In addition to these six functional receptors, a number of orphan receptors have been cloned from human and other species, that are structurally related to either CC- or CXC-chemokine receptors. These include the human BLR1 [13], EBI1 [5], LCR1 [21], the mouse MIP-1 RL1 and MIP-1 RL2 [17] and the bovine PPR1 [25]. Their respective ligand(s) and function(s) are unknown at present.

SUMMARY OF THE INVENTION

The present invention is related to a peptide having at least an amino acid sequence which presents more than 80%, advantageously more than 90%, preferably more than 95%, homology with the amino acid sequence as represented in SEQ ID NO. 1.

Preferably, said peptide has also at least an amino acid sequence which presents more than 80%, advantageously more than 90%, preferably more than 95%, homology with the amino acid sequence as represented in SEQ ID NO. 2.

According to another embodiment of the present invention, the peptide has at least an amino acid sequence which presents more than 80%, advantageously more than 90%, preferably more than 95%, homology with the amino acid sequence as represented in SEQ ID NO. 3.

The present invention is also related to the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or a portion thereof (represented in the FIG. 1).

A “portion of an amino acid sequence” means one or more amino acid segments having the same or improved binding properties of the whole peptide according to the invention. Said portion could be an epitope which is specifically binded by a ligand of the peptide which could be a known “natural ligand” of said peptide, an agonist or an analog of said ligand, or an inhibitor capable of competitively inhibiting the binding of said ligand to the peptide (including the antagonists of said ligand to the peptide).

Specific examples of said portions of amino acid sequence and their preparation process are described in the publication of Rucker J. et al. (Cell, Vol. 87, pp. 437-446 (1996)) incorporated herein by reference.

According to the invention, said portion of the amino acid sequence of the peptide according to the invention comprises the N-terminus segment and the first extracellular loop of the peptide.

Therefore, according to the invention, the amino acid sequence as represented in SEQ ID NO. 1 is the common amino acid sequence of SEQ ID NO. 2 and of SEQ ID NO. 3 (see also FIG. 1). Therefore, a first industrial application of said amino acid sequence is the identification of the homology between said amino acid sequence and the screening of various mutants encoding a different amino acid sequence than the one previously described, and the identification of various types of patient which may present a predisposition or a resistance to the disorders described in the following specification.

Preferably, the peptide according to the invention or a portion thereof is an active CC-chemokine receptor.

Advantageously, the CC-chemokine receptor according to the invention is stimulated by the MIP-1β chemokine at a concentration less or equal to 10 nm, and is advantageously also stimulated by the MIP-1α or RANTES chemokines. However, said chemokine receptor is not stimulated by the MCP-1, MCP-2, MCP-3, IL-8 and GROα chemokines.

In addition, the peptide according to the invention or a portion thereof is also a receptor of HIV viruses or a portion of said HIV viruses.

It is meant by “HIV viruses”, HIV-1 or HIV-2 and all the various strains of HIV viruses which are involved in the development of AIDS. It is meant by a “a portion of HIV viruses”, any epitope of said viruses which is able to interact specifically with said receptor. Among said portions of viruses which may be involved in the interaction with the peptide according to the invention, are peptides encoded by the ENV and GAG viruses genes.

Preferably, said portion of HTV viruses is the glycopeptide gp120/160 (membrane-bound gp160 or the free gp derived therefrom) or a portion thereof.

It is meant by a “portion of the glycopeptide gp120/160” any epitope, preferably an immuno-dominant epitope, of said glycopeptide which may interact specifically with the peptide according to the invention, such as for instance the V3 loop (third hypervariable domain).

According to another embodiment of the present invention, the peptide according to the invention is an inactive CC-chemokine receptor. An example of such inactive CC-chemokine receptor is encoded by the amino acid sequence as represented in SEQ ID NO. 2.

It is meant by an “inactive CC-chemokine receptor” a receptor which is not stimulated by any known CC-chemokine, especially the MIP-1β, MIP-1α or RANTES chemokines.

The peptide represented in SEQ ID NO. 3 according to the invention is an 30 inactive receptor which is not a receptor of HIV viruses or of a portion of said HIV viruses, which means that said inactive receptor does not allow the entry of said FHV viruses into a cell which presents at its surface said inactive receptor.

Advantageously, the peptide according to the invention is a human receptor.

The present invention concerns also the nucleic acid molecule having more than 80%, preferably more than 90%, homology with one of the nucleic acid sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 shown in the FIG. 1.

Preferably, said nucleic acid molecule has at least the nucleic acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 of FIG. 1 or a portion thereof.

It is meant by a “portion of said nucleic acid molecule” any nucleic acid sequence of more than 15 nucleotides which could be used in order to detect and/or reconstitute said nucleic acid molecule or its complementary strand. Such portion could be a probe or a primer which could be used in genetic amplification using the PCR, LCR, NASBA or CPR techniques for instance.

The present invention concerns more specifically the nucleic acid molecules encoding the peptide according to the invention. Said nucleic acid molecules are RNA or DNA molecules such as a cDNA molecule or a genomic DNA molecule.

The present invention is also related to a vector comprising the nucleic acid molecule according to the invention. Preferably, said vector is adapted for expression in a cell and comprises the regulatory elements necessary for expressing the amino acid molecule in said cell operatively linked to the nucleic acid sequence according to the invention as to permit expression thereof.

Preferably, said cell is chosen among the group consisting of bacterial cells, yeast cells, insect cells or mammalian cells. The vector according to the invention is a plasmid, preferably a pcDNA3 plasmid, or a virus, preferably a baculovirus, an adenovirus or a semliki forest virus.

The present invention concerns also the cell, preferably a mammalian cell, such as a CHO-K1 or a HEK293 cell, transformed by the vector according to the invention. Advantageously, said cell is non neuronal in origin and is chosen among the group consisting of CHO-K1, HEK293, BHK21, COS-7 cells.

The present invention also concerns the cell (preferably a mammalian cell such as a CHO-K1 cell) transformed by the vector according to the invention and by another vector encoding a protein enhancing the functional response in said cell. Advantageously, said protein is the Gα15 or Gα16 (G protein, α subunit). Advantageously, said cellis the cell CHO-K1-pEFIN hCCR5-1/16.

The present invention is also related to a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of the nucleic acid molecule according to the invention. Said nucleic acid probe may be a DNA or a RNA.

The invention concerns also an antisense oligonucleotide having a sequence capable of specifically hybridizing to an MRNA molecule encoding the peptide according to the invention so as to prevent translation of said MIRNA molecule or an antisense oligonucleotide having a sequence capable of specifically hybridizing to the CDNA molecule encoding the peptide according to the invention.

Said antisense oligonucleotide may comprise chemical analogs of nucleotide or substances which inactivate MRNA, or be included in an RNA molecule endowed with ribozyme activity.

Another aspect of the present invention concerns a ligand or an anti-ligand (preferably an antibody) other than known “natural ligands”, which are chosen among the group consisting of the MIP-1β, MIP-1α or RANTES chemokines, HIV viruses or a portion of said HIV viruses, wherein said ligand is capable of binding to the receptor according to the invention and wherein said anti-ligand is capable of (preferably competitively) inhibiting the binding of said known “natural ligand” or the ligand according to the invention to the peptide according to the invention.

The exclusion in the above identified definition of known chemokines, HIV viruses or a portion of said HIV viruses, does not include variants of said “natural” viruses or said “natural” portion which may be obtained for instance by genetic engineering and which may mimic the interaction of said viruses and portion of said viruses to the peptide according to the invention.

Advantageously, said antibody is a monoclonal antibody which is preferably directed to an epitope of the peptide according to the invention and present on the surface of a cell expressing said peptide.

Preferably, said antibody is produced by the hybridome cell AchCCR5-SAB1A7.

The invention concerns also the pharmaceutical composition comprising either an effective amount of the peptide according to the invention (in order to delude the HIV virus from the natural peptide present at the surface of a mammalian cell and stop the infection of said mammalian cell by the HIV virus), or an effective amount of the above identified described ligand and/or anti-ligand, or an effective amount of oligonucleotide according to the invention, effective to decrease the activity of said peptide by passing through a cell membrane and binding specifically with MRNA encoding the peptide according to the invention in the cell so as to prevent it translation. The pharmaceutical composition comprises also a pharmaceutically acceptable carrier, preferably capable of passing through said cell membrane.

Preferably, in said pharmaceutical composition, the oligonucleotide is coupled to a substance, such as a ribozyme, which inactivates MRNA encoding the peptide according to the invention.

Preferably, the pharmaceutically acceptable carrier comprises a structure which binds to a receptor on a cell capable of being taken up by cell after binding to the structure. The structure of the pharmaceutically acceptable carrier in said pharmaceutical composition is capable of binding to a receptor which is specific for a selected cell type.

The present invention concerns also a transgenic non human mammal overexpressing (or expressing ectopically) the nucleic acid molecule encoding the peptide according to the invention.

The present invention also concerns a transgenic non human mammal comprising an homologous recombination knockout of the native peptide according to the invention.

According to a preferred embodiment of the invention, the transgenic non human mammal whose genome comprises antisense nucleic acid complementary to the nucleic acid according to the invention is so placed as to be transcripted into antisense MRNA which is complementary to the MRNA encoding the peptide according to the invention and which hybridizes to MRNA encoding said peptide, thereby reducing its translation. Preferably, the transgenic non human mammal according to the invention comprises a nucleic acid molecule encoding the peptide according to the invention and comprises additionally an inducible promoter or a tissue specific regulatory element.

Preferably, the transgenic non human mammal is a mouse.

The invention relates to a method for determining whether a ligand can be specifically bound to the peptide according to the invention, which comprises contacting a cell transfected with a vector expressing the nucleic acid molecule encoding said peptide with the ligand under conditions permitting binding of ligand to such peptide and detecting the presence of any such ligand bound specifically to said peptide, thereby determining whether the ligand binds specifically to said peptide.

The invention relates to a method for determining whether a ligand can specifically bind to a peptide according to the invention, which comprises preparing a cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said peptide, isolating a membrane fraction from the cell extract, contacting the ligand with the membrane fraction under conditions permitting binding of the ligand to such peptide and detecting the presence of any ligand bound to said peptide, thereby determining whether the compound is capable of specifically binding to said peptide. Preferably, said method is used when the ligand is not previously known.

The invention relates to a method for determining whether a ligand is an agonist of the peptide according to the invention, which comprises contacting a cell transfected with a vector expressing the nucleic acid molecule encoding said peptide with the ligand under conditions permitting the activation of a functional peptide response from the cell and detecting by means of a bio-assay, such as a modification in a second messenger concentration (preferably calcium ions or inositol phosphates such as IP₃) or a modification in the cellular metabolism (preferably determined by the acidification rate of the culture medium), an increase in the peptide activity, thereby determining whether the ligand is a peptide agonist.

The invention relates to a method for determining whether a ligand is an agonist of the peptide according to the invention, which comprises preparing a cell extract from cells transfected with a vector expressing the nucleic acid molecule encoding said peptide, isolating a membrane fraction from the cell extract, contacting the membrane fraction with the ligand under conditions permitting the activation of a functional peptide response and detecting by means of a bio-assay, such as a modification in the production of a second messenger (preferably inositol phosphates such as IP₃), an increase in the peptide activity, thereby determining whether the ligand is a peptide agonist.

The present invention relates to a method for determining whether a ligand is an antagonist of the peptide according to the invention, which comprises contacting a cell transfected with a vector expressing the nucleic acid molecule encoding said peptide with the ligand in the presence of a known peptide agonist, under conditions permitting the activation of a functional peptide response and detecting by means of a bio-assay, such as a modification in second messenger concentration (preferably calcium ions or inositol phosphates such as IP₃) or a modification in the cellular metabolism (preferably determined by the acidification rate of the culture medium), a decrease in the peptide activity, thereby determining whether the ligand is a peptide antagonist.

The present invention relates to a method for determining whether a ligand is an antagonist of the peptide according to the invention, which comprises preparing a cell extract from cells transfected with an expressing the nucleic acid molecule encoding said peptide, isolating a membrane fraction from the cells extract, contacting the membrane fraction with the ligand in the presence of a known peptide agonist, under conditions permitting the activation of a functional peptide response and detecting by means of a bio-assay, such as a modification in the production of a second messenger, a decrease in the peptide activity, thereby determining whether the ligand is a peptide antagonist.

Preferably, the second messenger assay comprises measurement of calcium ions or inositol phosphates such as IP₃.

Preferably, the cell used in said method is a mammalian cell non neuronal in origin, such as CHO-K1, HEK293, BHK21, COS-7 cells.

In said method, the ligand is not previously known.

The invention is also related to the ligand isolated and detected by any of the preceding methods.

The present invention concerns also the pharmaceutical composition which comprises an effective amount of an agonist or an antagonist of the peptide according to the invention, effective to reduce the activity of said peptide and a pharmaceutically acceptable carrier.

It is meant by “an agonist or an antagonist of the peptide according to the invention”, all the agonists or antagonists of the known “natural ligand” of the peptide as above described.

Therefore, the previously described methods may be used for the screening of drugs to identify drugs which specifically bind to the peptide according to the invention.

The invention is also related to the drugs isolated and detected by any of these methods.

The present invention concerns also a pharmaceutical composition comprising said drugs and a pharmaceutically acceptable carrier.

The invention is also related to a method of detecting expression of a peptide according to the invention by detecting the presence of MRNA coding for a peptide, which comprises obtaining total RNA or total MRNA from the cell and contacting the RNA or MRNA so obtained with the nucleic acid probe according to the invention under hybridizing conditions and detecting the presence of MRNA hybridized to the probe, thereby detecting the expression of the peptide by the cell.

Said hybridization conditions are stringent conditions.

The present invention concerns also the use of the pharmaceutical composition according to the invention for the treatment and/or prevention of inflammatory diseases, including rheumatoid arthritis, glomerulonephritis, asthma, idiopathic pulmonary fibrosis and psoriasis, viral infections including Human Immunodeficiency Viruses 1 and 2 (HIV-1 and 2), cancer including leukaemia, atherosclerosis and/or auto-immune disorders.

The present invention concerns also a method for diagnosing a predisposition or a resistance to a disorder associated with the activity of the peptide according to the invention and/or associated with infectious agents such as HIV viruses in a subject. Said method comprises

a) obtaining nucleic acid molecules encoding the peptide according to the invention from the cells of the subject;

b) possibly performing a restriction digest of said nucleic acid molecules with a panel of restriction enzymes;

c) possibly electrophoretically separating the resulting nucleic acid fragments on a sized gel;

d) contacting the resulting gel or the obtained nucleic acid molecule with a nucleic acid probe labelled with a detectable marker and capable of specifically hybridizing to said nucleic acid molecule (said hybridization being made in stringent hybridization conditions);

e) detecting labelled bands or the in situ nucleic acid molecules which have hybridized to the said nucleic acid molecule labelled with a detectable marker to create a unique band pattern or an in situ marking specific to the subject;

f) preparing other nucleic acid molecules encoding the peptide according to the invention obtained from the cells of other patients for diagnosis by step a-e; and

g) comparing the unique band pattern specific to the nucleic acid molecule of subjects suffering from the disorder from step e and the nucleic acid molecule obtained for diagnosis from step f to determine whether the patterns are the same or different and to diagnose thereby a predisposition or a resistance to the disorder if the patterns are the same or different.

The present invention is also related to a method for diagnosing a predisposition or a resistance to a disorder associated with the activity of a specific allele of the peptide according to the invention or the presence of said peptide at the surface of cells and/or: associated with infectious agents such as HIV viruses present in a subject. Said method comprises:

a) obtaining a sample of a body fluid, preferably a blood sample comprising antigen presenting cells, from a subject;

b) adding to said sample a ligand and/or an anti-ligand according to the invention;

c) detecting the cross-reaction between said ligand and/or said anti-ligand and the specific peptide according to the invention; and

d) determining whether the peptide corresponds to a receptor or an inactive receptor according to the invention and diagnosing thereby a predisposition or a resistance to the disorder according to the type of the peptide present in the body fluid of the subject.

The present invention concerns also a diagnostic and/or dosage device, preferably a kit, comprising the peptides, the nucleic acid molecules, the nucleic acid probes, the ligands and/or the anti-ligands according to the invention, their portions (such as primers, probes, epitopes, . . . ) or a mixture thereof, being possibly labelled with a detectable marker.

Said diagnostic and/or dosage device comprises also the reactants for the detection and/or the dotage of antigens, antibodies or nucleic acid sequences through a method selected from the group consisting of in situ hybridization, hybridization or recognition by marked specific antibodies, specially ELISA® (Enzyme Linked Immunosorbent Assay) or RIA® (Radio Immunoassay), methods on filter, on a solid support, in solution, in “sandwich”, on gel, by Dot blot hybridization, by Northern blot hybridization, by Southern blot hybridization, by isotopic or non-isotopic labelling (such as immunofluorescence or biotinylation), by a technique of cold probes, by genetic amplification, particularly PCR, LCR, NASBA or CPR, by a double immunodiffusion, by a counter-immunoelectrophoresis, by haemagglutination and/or a mixture thereof.

A last aspect of the present invention concerns a method of preparing peptides according to the invention, which comprises

a) constructing a vector adapted for expression in a cell which comprises the regulatory elements necessary for the expression of nucleic acid molecules in the cell operatively linked to nucleic acid molecule encoding said peptide so as to permit expression thereof, wherein the cell is preferably selected from the group consisting of bacterial cells, yeast cells, insect cells and mammalian cells;

b) inserting the vector of step a in a suitable host cell;

c) incubating the cell of step b under conditions allowing the expression of the peptide according to the invention;

d) recovering the peptide so obtained; and

e) purifying the peptide so recovered, thereby preparing an isolated peptide -according to the invention.

The deposits of micro-organisms AchCCR5-SAB1A7 and CHO-K1-PEFIN HCCR5-1/16 were made according to the Budapest Treaty in the Belgium Coordinated Collection of Micro-organisms (BCCM), Laboratorium voor Moleculaire Biologic (LMBP), Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 GENT, BELGIUM.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Shows the nucleic acid and amino acid sequences of the invention.

FIGS. 1A-1 and 1A-2 show the nucleic acid and amino acid sequence of SEQ ID Nos 1 and 4, respectively.

FIGS. 1B-1, 1B-2, and 1B-3 show the nucleic acid and amino acid sequence of SEQ ID Nos 2 and 5, respectively.

FIGS. 1D-1 to 1D-3 show the nucleic acid and amino acid sequence of SEQ ID Nos. 3 and 6, respectively.

FIG. 2 represents the amino acids sequence of the active human CCR5 chemokine receptor (SEQ ID NO: 5) according to the invention aligned with that of the human CCR1 (SEQ ID NO: 9), CCR2b (SEQ ID NO: 7), CCR3 (SEQ ID NO: 8), and CCR4 (SEQ ID NO: 10) receptors. Amino acids identical with the active CCR5 sequence are boxed.

FIG. 3 shows the chromosomal organisation of the human CCR2 and CCR5 chemokine receptor genes.

FIG. 4 shows the functional expression of the human active CCR5 receptor in a CHO-K1 cell line.

FIG. 5 represents the distribution of MRNA encoding the CCR5 receptor in a panel of human cell lines of haematopoietic origin.

FIG. 6 represents the structure of the mutant form of human CCR5 receptor. FIG. 6B shows the wild type amino acid sequence (SEQ ID NO: 11), and the location of the 32 base deletion mutation in the nucleic acid (SEQ ID NO: 12) and amino acid sequences (SEQ ID NO: 13).

FIG. 7 represents the quantification of ENV proteins-mediated fusion by luciferase assays.

FIG. 8 represents genotyping of individuals by PCR and segregation of the CCR5 alleles in CEPH families.

FIG. 9 represents the FACS analysis of sera anti-CCR5 on a CCR5-CHO cell line according to the invention.

FIG. 10 represents the inhibition of HIV infectivity with anti-CCR5 antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

1. Experimentals

Materials

Recombinant human chemokines, including MCP-1, MIP-1α, MIP-1β, RANTES, IL-8 and GROα were obtained from R & D Systems (London, UK). [¹²⁵I]MIP-1α (specific activity, 2200 Ci/mmol) was obtained from Dupont NEN (Brussels, Belgium). Chemokines obtained from R & D Systems were reported by the supplier as >97% pure on SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and biologically active on a bioassay specific for each ligand. The lyophilised chemokines were dissolved as a 100 μg/ml solution in a sterile phosphate-buffered saline (PBS) and this stock solution was stored at −20° C. in aliquots. Chemokines were diluted to the working concentration immediately before use. All cell lines used in the present study were obtained from the ATCC (Rockville, Md., USA).

Cloning and Sequencing

The mouse MOP020 clone was obtained by low stringency polymerase chain reaction, as described previously [24, 34], using genomic DNA as template. A human genomic DNA library (Stratagene, La Jolla, Calif.), constructed in the lambda DASH vector was screened at low stringency [39] with the MOP020 (511 bp) probe. The positive clones were purified to homogeneity and analysed by Southern blotting. The restriction map of the locus was determined and a relevant XbaI fragment of 4,400 bp was subcloned in pBluescript SK+ (Stratagene). Sequencing was performed on both strands after subcloning in M13mp derivatives, using fluorescent primers and an automated DNA sequencer (Applied Biosystem 370A). Sequence handling and data analysis was carried out using the DNASIS/PROSIS software (Hitachi), and the GCG software package (Genetics Computer Group, Wisconsin).

Expression in Cell Lines

The entire coding region was amplified by PCR as a 1056 bp fragment, using primers including respectively the BamHI and XbaI recognition sequences, and cloned after restriction in the corresponding sites of the eukaryotic expression vector pcdna3 (Invitrogen, San Diego, Calif.). The resulting construct was verified by sequencing, and transfected in CHO-K1 cells as described [35]. Two days after transfection, selection for stably transfected cell lines was initiated by the addition of 400 μg/ml G418 (Gibco), arid resistant clones were isolated at day 10. CHO-K1 cells were cultured using Ham's F12 medium, as previously described [35, 11]. The expression of the active CCR5 receptor in the various cell clones was evaluated by measuring the specific transcript level by Northern blotting, on total RNA prepared from the cells (see below).

Binding Assays

Stably transfected CHO-K1 cells expressing the active CCR5 receptor were grown to confluence and detached from culture dishes by incubation in phosphate-buffered saline (PBS) supplemented with 1 Mm EDTA. Cells were collected by low speed centrifugation and counted in a Neubaeur cell. Binding assays were performed in polyethylene minisorp tubes (Nunc) in a final volume of 200 μl PBS containing 0.2% bovine serum albumin (BSA) and 10⁶ cells, in presence of [¹²⁵I]-MIP-1α. Non specific binding was determined by addition of 10 Nm unlabelled MIP-1α. The concentration of labelled ligand was 0.4 Nm (around 100,000 cpm per tube). The incubation was carried out for 2 hours at 4° C., and was: stopped by the rapid addition of 4 ml ice-cold buffer, and immediate collection of cells by vacuum filtration through GF/B glass fiber filters (Whatmann) pre-soaked in 0.5% polyethyleneinimine (Sigma). Filters were washed three times with 4 ml ice-cold buffer and counted in a gamma counter.

Biological Activity

The CHO-K1 cell lines stably transfected with the pcdna3/CCR5 construct or wild type CHO-K1 cells (used as controls) were plated onto the membrane of Transwell cell capsules (Molecular Devices), at a density of 2.5 10⁵ cells/well in Ham's F12 medium. The next day, the capsules were transferred in a microphysiometer (Cytosensor, Molecular Devices), and the cells were allowed to equilibrate for approximately two hours by perfusion of 1 Mm phosphate-buffered (Ph 7.4) RPMI-1640 medium containing 0.2% BSA. Cells were then exposed to various chemokines diluted in the same medium, for a 2 mm duration. Acidification rates were measured at one minute intervals.

Northern Blotting

Total RNA was isolated from transfected CHO-K1 cell lines, from a panel of human cell lines of haematopoietic origin and from a panel of dog tissues, using the RNeasy kit (Qiagen). RNA samples (10 μg per lane) were denatured in presence of glyoxal [26], fractionated on a 1% agarose gel in a 10 Mm phosphate buffer (Ph 7.0), and transferred to nylon membranes (Pall Biodyne A, Glen Cove, N.Y.) as described [42]. After baking, the blots were prehybridized for 4 h at 42° C. in a solution consisting of 50% formamide, 5×Denhardt solution (1×Denhardt: 0.02% Ficoll, 0.02% polyvinylpyrolidone, 0.02% BSA), 5×SSPE (1×SSPE: 0.18 M NaCl, 10 Mm Na phosphate, 1 Mm EDTA Ph 8.3), 0.3% Sodium Dodecyl Sulphate (SDS), 250 μg per ml denatured DNA from herring testes. DNA probes were (α³²P)-labelled by random priming [14]. Hybridizations were carried out for 12 h at 42° C. in the same solution containing 10% (wt/vol) dextran sulphate and the heat denatured probe. Filters were washed up to 0.1×SSMC (1×SSC: 150 Mm NaCl, 15 Mm Na Citrate Ph 7.0), 0.1% SDS at 60° C. and autoradiographed at −70° C. using Amersham β-max films.

2. Results and Discussion

Cloning and Structural Analysis

The sequence homology characterising genes encoding G protein-coupled receptors has allowed the cloning by low stringency polymerase chain reaction (PCR) of new members of this gene family [24, 34]. One of the clones amplified from mouse genomic DNA, named MOP020 presented strong similarities with characterised chemokine receptors, sharing 80% identity with the MCP-1 receptor (CCR2) [8], 65% identity with the MIP-1α/RANTES receptor (CCR1) [31], and 51% identity with IL-8 receptors [20, 30]. The clone was used as a probe to screen a human genomic library. A total of 16 lambda phage clones were isolated. It was inferred from the restriction pattern of each clone and from partial sequence data that all clones were belonging to a single contig in which two different coding sequences were included. One of the coding sequences was identical to the reported CDNA encoding the CCR2 receptor [8, 44]. A 4.400 pb XbaI fragment of a representative clone containing the second region of hybridization was subcloned in Pbluescript SK+. Sequencing revealed a novel gene, tentatively named CCR5, sharing 84% identity with the MOP020 probe, suggesting that MOP020 is the mouse ortholog of CCR5. MOP020 does not correspond to any of the three mouse chemokine receptor genes cloned recently [16], demonstrating the existence of a fourth murine chemokine receptor.

The sequence of CCR5 revealed a single open reading frame of 352 codons encoding a protein of 40,600 Da. The sequence surrounding the proposed initiation codon is in agreement with the consensus as described by Kozak [22], since the nucleotide in −3 is a purine. The hydropathy profile of the deduced amino acid sequence is consistent with the existence of 7 transmembrane segments. Alignment of the CCR5 amino acid sequence with that of other functionally characterised human CC-chemokine receptors is represented in FIG. 2. The highest similarity is found with the CCR2 receptor [8] that shares 75.8% identical residues. There is also 56.3% identity with the CCR1 receptor [31], 58.4% with the CCR3 [10], and 49.1% with the CCR4 [37]. CCR5 represents therefore a new member of the CC-chemokine receptor group [30]. Like the related CCR1 and IL-8 receptors [20, 29, 31, 16] the coding region of CCR5 appears as intronless. From our partial sequencing data, the CCR2 gene is also devoid of intron in the first two thirds of its coding sequence.

Sequence similarities within the chemokine receptor family are higher in the transmembrane-spanning domains, and in intracellular loops. As an example, the identity score between CCR5 and CCR2 goes up to 92% when considering the transmembrane segments only. Lower similarities are found in the N-terminal extracellular domain, and in the extracellular loops. The N-terminal domain of the IL-8 and CCR2 receptors has been shown to be essential for interaction with the ligand [19, 18]. The variability of this region among CC-chemokine receptors presumably contributes to the specificity towards the various ligands of the family.

A single potential site for N-linked glycosylation was identified in the third extracellular loop of CCR5 (FIG. 1). No glycosylation site was found in the N-terminal domain of the receptor, where most G protein-coupled receptors are glycosylated. The other chemokine receptors CCR1 and CCR2 present such an N-linked glycosylation site in their N-terminal domain [31, 8]. By contrast, the CCR3 receptor [10] does not display glycosylation sites neither in the N-terminus, nor in extracellular loops. The active CCR5 receptor has four cysteines in its extracellular segments, and all four are conserved in the other CC- and CXC-chemokine receptors (FIG. 2). The cysteines located in the first and second extracellular loops are present in most G protein-coupled receptors, and are believed to form a disulphide bridge stabilising the receptor structure [41]. The two other cysteines, in the N-terminal segment, and in the third extracellular loop could similarly form a stabilising bridge specific to the chemokine receptor family. The intracellular domains of CCR5 do not include potential sites for phosphorylation by protein kinase C (PKC) or protein kinase A. PKC sites, involved in heterologous desensitisation are frequent in the third intracellular loop and C-terminus of G protein-coupled receptors. CCR1 is also devoid of PKC sites. In contrast, all CC-chemokine receptors, are rich in serine and threonine residues in the C-terminal domain. These residues represent potential phosphorylation sites by the family of G protein-coupled receptor kinases, and are probably involved in homologous desensitisation [41]. Five of these S/T residues are perfectly aligned in all five receptors (FIG. 2).

Physical Linkage of the CCR5 and CCR2 Genes

As stated above, the 16 clones isolated with the MOP020 probe corresponded to a single contig containing the CCR5 and CCR2 genes. The organisation of this contig was investigated in order to characterise the physical linkage of the two receptor genes in the human genome. A combination of restriction mapping, Southern blotting, fragment subcloning and partial sequencing allowed to determine the respective borders and overlaps of all clones. Out of the 16 clones, 9 turned out to be characterised by a specific restriction map, and their organisation is depicted in FIG. 3. Four of these clones (#11, 18, 21, 22) contained the CCR2 gene alone, four clones (# 7, 13, 15, 16) contained the ChemR13 gene alone and one clone (#9) contains part of both coding sequences. The CCR2 and CCR5 genes are organised in tandem, CCR5 being located downstream of CCR2. The distance separating CCR2 and CCR5 open reading frames is 17.5 kb. The chromosomal localisation of the tandem is presently unknown. Other chemokine receptors have however been located in the human genome: the CCR1 gene was localised by fluorescence in situ hybridization to the p21 region of human chromosome 3 [16]. The two IL-8 receptor genes, and theft pseudogene have been shown to be clustered on the human 2q34-q35 region [1].

Functional Expression and Pharmacology of the Active CCR5 Receptor

Stable CHO-K1 cell lines expressing the active CCR5 receptor were established and were screened on the basis of the level of CCR5 transcripts as determined by Northern blotting. Three clones were selected and tested for biological responses in a microphysiometer, using various CC- and CXC-chemokines as potential agonists. Wild type CHO-K1 dells were used as control to ensure that the observed responses were specific for the transfected receptor, and did not result from the activation of endogenous receptors. The microphysiometer allows the real time detection of receptor activation, by measuring the modifications of cell metabolism resulting from the stimulation of intracellular cascades [33]. Several studies have already demonstrated the potential of microphysiometry in the field of chemokine receptors. Modifications of metabolic activity in human monocytes, in response CC-chemokines, were monitored using this system [43]. Similarly, changes in the acidification rate of THP-1 cells (a human monocytic cell line) in response to MCP-1 and MCP-3 have been measured [36]. The estimation of the EC₅₀ for both proteins, using this procedure, was in agreement with the values obtained by monitoring the intracellular calcium in other studies [8, 15].

Ligands belonging to the CC- and CXC-chemokine classes were tested on the CCR5 transfected CHO-K1 cells. Whereas MIP-1α, MIP-1α and RANTES were found to be potent activators of the new receptor (FIG. 4), the CC-chemokines MCP-1, MCP-2 and MCP-3, and the CXC-chemokines GROα and IL-8 had no effect on the metabolic activity, even at the highest concentrations tested (30 Nm). The biological activity of one of the chemokines inducing no response on CCR5 (IL-8) could be demonstrated on a CHO-K1 cell line transfected with the IL-8A interleukin receptor (Mollereau et al., 1993): IL-8 produced a 160% increase in metabolic activity as determined using the microphysiometer. The biological activity of the MCP-2 and MCP-3 preparations as provided by J. Van Damme have been widely documented [2, 40]. MIP-1α, MIP-1β and RANTES were tested on the wild type CHO-K1 cells, at a 30 Nm-concentration, and none of them induced a metabolic response. On the CCR5 transfected CHO-K1 cell line, all three active ligands (MIP-1α, MIP-1β and RANTES) caused a rapid increase in acidification rate, reaching a maximum by the second or third minute after perfusion of the ligand. The acidification rate returned to basal level within 10 minutes. The timing of the cellular response is similar to that observed for chemokines on their natural receptors in human monocytes [43]. When agonists were applied repeatedly to the same cells, the response was strongly reduced as compared to the first stimulation, suggesting the desensitisation of the receptor. All measurements were therefore obtained on the first stimulation of each capsule.

The concentration-effect relation was evaluated for the three active ligands in the 0.3 to 30 Nm range (FIGS. 3B and C). The rank order of potency was MIP-1α>MIP-1β=RANTES. At 30 Nm concentrations, the effect of MIP-1α appeared to saturate (at 156% of baseline level) while MIP-1β and RANTES were still in the ascending phase. Higher concentrations of chemokines could however not be used. The EC50 was estimated around 3 Nm for MIP-1α. The concentrations necessary for obtaining a biological response as determined by using the microphysiometer are in the same range as those measured by intracellular calcium mobilisation for the CCR1 [31], the CCR2A and B [8], and the CCR3 [10] receptors. The ligand specificity of CCR5 is similar to that reported for CCR3 [10]. CCR3 was described as the first cloned receptor responding to MIP-1β. However, MIP-1β at 10 Nm elicits a significant effect on the CCR5, while the same concentration is without effect on the CCR3 transfected cells [10]. These data suggest that CCR5 could be a physiological receptor for MIP-1β.

Binding experiments using [¹²⁵I]-human MIP-1α as ligand did not allow to demonstrate specific binding to CCR53 expressing CHO-K1 cells, using as much as 0.4 Nm radioligand and 1 million transfected cells per tube. Failure to obtain binding data could be attributed to a relatively low affinity of the receptor for MIP-1α.

Northern Blotting Analysis

Northern blotting performed on a, panel of dog tissues did not allow to detect transcripts for CCR5. Given the role of the chemokine receptor family in mediating chemoattraction and activation of various classes of cells involved in inflammatory and immune responses, the probe was also used to detect specific transcripts in a panel of human cell lines of haematopoietic origin (FIG. 5). The panel included lymphoblastic (Raji) and T lymphoblastic (Jurkat) cell lines, promyeloblastic (KG-1A) and promyelocytic (HL-60) cell lines, a monocytic (THP-1) cell line, an erythroleukemia (HEL 92.1.7) cell line, a megakaryoblastic (MEG-01) cell line, and a myelogenous leukaemia (K-562) cell line. Human peripheral blood mononuclear cells (PBMC), including mature monocytes and lymphocytes, were also tested. CCR5 transcripts (4.4 kb) could be detected only in the KG-1A promyeloblastic cell line, but were not found in the promyelocytic cell line HL-60, in PBMC, or in any of the other cell lines tested. These results suggest that the active CCR5 receptor could be expressed in precursors of the granulocytic lineage. CC-chemokines have been reported to stimulate mature granulocytes [27, 38, 23, 2]. However, recent data have also demonstrated a role of CC- and CXC-chemokines in the regulation of mouse and human myeloid progenitor cell proliferation [6, 7].

CCR5 was shown to respond to MIP-1α, MIP-1β and RANTES, the three chemokines identified as the major HIV-suppressive factors produced by CD8⁺ T cells [9], and released in higher amounts by CD4⁺ T lymphocytes from uninfected but multiply exposed individuals [51]. CCR5 represents a major co-receptor for macrophage-tropic (M-tropic) HIV-1 primary isolates and strains [45, 50]. M-tropic strains predominate during the asymptomatic phase of the disease in infected individuals, and are considered as responsible for HIV-1 transmission. Strains adapted for growth in transformed T-cell lines (T-tropic strains) use as a co-receptor LESTR (or fusin) [50], an orphan receptor also belonging to the chemokine receptor family, but not yet characterized functionally [21, 52, 53]. Dual-tropic viruses, which may represent transitional forms of the virus in late stages of infection [54] are shown to use both CCR5 and LESTR as co-receptors, as well as the CC-chemokine receptors CCR2b and CCR3 [47]. The broad spectrum of co-receptor usage of dual-tropic viruses suggests that within infected individuals, the virus may evolve at least in part from selection by a variety of co-receptors expressed on different cell types.

Identification of an Inactive ΔCCR5 Receptor

It is known that some individuals remain uninfected despite repeated exposure to HIV-1 [55, 56, 51]. A proportion of these exposed-uninfected individuals results from the relatively low risk of contamination after a single contact with the virus, but it has been postulated that truly resistant individuals do exist. In fact, CD4⁺ lymphocytes isolated from exposed-uninfected individuals are highly resistant to infection by primary M-tropic, but not T-tropic HIV-1 strains. Also, peripheral blood mononuclear cells (PBMC) from different donors are not infected equally with various HIV-1 strains [57-59]. Given the key role played by CCR5 in the fusion event that mediates infection by M-tropic viruses, it is postulated that variants of CCR5 could be responsible for the relative or absolute resistance to HIV-1 infection exhibited by some individuals, and possibly for the variability of disease progression in infected patients [66]. The Inventors selected three HIV-1 infected patients known to be slow progressors, and four seronegative individuals as controls; the full coding region of their CCR5 gene was amplified by PCR and sequenced. Unexpectedly, one of the slow progressors, but also two of the uninfected controls, exhibited heterozygosity at the CCR5 locus for a biallelic polymorphism. The frequent allele corresponded to the published CCR5 sequence, while the minor one displayed a 32 bp deletion within the coding sequence, in a region corresponding to the second extracellular loop of the receptor (FIG. 6). The FIG. 6 is the structure of the mutant form of human CC-chemokine receptor 5. a. The amino acid sequence of the non-functional Δccr5 protein is represented. The transmembrane organisation is given by analogy with the predicted transmembrane structure of the wild-type CCR5. Amino acids represented in black correspond to unnatural residues resulting from the frame shift caused by the deletion. The mutant protein lacks the last three transmembrane segments of CCR5, as well as the regions involved in G protein-coupling b, Nueleotide sequence of the CCR5 gene surrounding the deleted region, and translation into the normal receptor (top) or the truncated mutant (ccr5, bottom). The 10-bp direct repeat is represented in italics. The full size .coding region of the CCR5 gene was amplified by PCR, using 5′-TCGAGGATCCAAGATGGATTATCAAGT-3′ (SEQ ID NO: 14) and 5′-CTGATCTAGAGCCATGTGCACAACTCT-3′ (SEQ ID NO: 15) as forward and reverse primers' respectively. The PCR products were sequenced on both strands using the same oligonucleotides as primers, as well as internal primers, and fluorochrome-labelled didcoxynucleotides as terminators. The sequencing products were run on an Applied Biosystem sequencer, and ambiguous positions were searched along the coding sequence. When the presence of a deletion was suspected from direct sequencing, the PCR products were cloned after restriction with BamHI and XbaI endonucleases into pcdna3. Several clones were sequenced to confirm the deletion. The deletion was identical in three unrelated individuals investigated by sequencing.

Cloning of the PCR product and sequencing of several clones confirmed the deletion. The deletion causes a frame shift, which is expected to result in premature termination of translation. The protein encoded by this mutant allele (Δccr5) therefore lacks the last three transmembrane segments of the receptor. A 10-bp direct repeat flanking the deleted region (FIG. 6b) on both sides is expected to have promoted the recombination event leading to the deletion. Numerous mutagenesis studies performed on various classes of G protein-coupled receptors, including chemokine receptors, makes it clear that such a truncated protein is certainly not functional in terms of chemokine-induced signal transduction: it lacks the third intracellular loop and C-terminal cytoplasmic domains, the two regions involved primarily in G protein coupling [41]. In order to test whether the truncated protein was able to function as a HIV-1 co-receptor, the Inventors tested its ability to support membrane fusion by both primary M-tropic and dual-tropic virus ENV proteins. The recombinant protein was expressed in quail QT6 cells together with human CD4. The QT6 cells were then mixed with HeLa cells expressing the indicated viral ENV protein and the extent of cell—cell fusion measured using a sensitive and quantitative gene-reporter assay. In contrast to wild-type CCR5, the truncated receptor did not allow fusion with cells expressing the ENV protein from either M-tropic or dual-tropic viruses (FIG. 7). The FIG. 7 represents the quantification of ENV protein-mediated fusion by luciferase assay. To quantify cell—cell fusion events, Japanese quail QT6 fibrosarcoma cells were transfected or cotransfected as indicated with the pcdna3 vector (Invitrogen) containing the coding sequence for wild-type CCR5, the truncated ccr5 mutant, the CCR2b or the Duffy chemokine receptors, or with the PCDNA3 vector alone. The target cells were also transfected with human CD4 expressed from the CMV promoter and the luciferase gene under the control of the T7 promoter. HeLa effector cells were infected (MOI=10) with vaccinia vectors expressing T7-polymerase (vTF1.1) and either the JR-FL (vCB28) or 89.6 (vBD3) envelope proteins. The luciferase activity resulting from cell fusion is expressed as the percentage of the activity (in relative light units) obtained for wild-type CCR5. All transfections were performed with an identical quantity of plasmid DNA using pcdna3 as carrier when necessary. To initiate fusion, target and effector cells were mixed in 24 well plates at 37° C. in the presence of ara-C and rifampicin, and allowed to fuse for 8 hours. Cells were lysed in 150 μl of reporter lysis buffer (Promega) and assayed for luciferase activity according to the manufacturer's instructions (Promega).

Coexpression of Δccr5 with wild-type CCR5 consistently reduced the efficiency of fusion for both JR-FL and 89.6 envelopes, as compared with CCR5 alone. Whether this in vitro inhibitory effect (not shared by the chemokine receptor Duffy, used as control) also occurs in vivo is presently not known. Coexpression with the CCR2b receptor [31], which is the CC-chemokine receptor most closely related to CCR5 but does not promote fusion by M-tropic HIV-1 strains [48], did not rescue the mutation by formation of a hybrid molecule (FIG. 7).

The FIG. 8 represents genotyping of individuals by PCR and segregation of the CCR5 alleles in CEPH families, α, Autoradiography illustrating the pattern resulting from PCR amplification and EcoRI cleavage for individuals homozygous for the wild-type CCR5 allele (CCR5/CCR5), the null Δccr5 allele (Δccr5/Δccr5), and for heterozygotes (CCR5/Δccr5). A 735 bp PCR product is cleaved into a common band of 332 bp for both alleles, and into 403 and 371 bp bands for the wild-type and mutant alleles, respectively. b, Segregation of the CCR5 alleles in two informative families of the CEPH. Half-black and white symbols represent heterozygotes and wild-type homozygotes, respectively. For a few individuals in the pedigrees, DNA was not available (ND: not determined). PCRs were performed on genomic DNA samples, using 5′-CCTGGCTGTCGTCCATGCTG-3′ (SEQ ID NO: 16) and 5′-CTGATCTAGAGCCATGTGCACAACTCT-3′ (SEQ ID NO: 17) as forward and reverse primers respectively. Reaction mixtures consisted in 30 μl of 10 Mm Tris-Hcl buffer Ph 8.0, containing 50 Mm Kcl, 0.75 Mm MgCl₂, 0.2 Mm dCTP, dGTP and dTTP, 0.1 Mm dATP, 0.5 μi [a-³²P]-DATP, 0.01% gelatine, 5% DMSO, 200 ng target DNA, 60 ng of each of the primers and 1.5 U Taq polymerase. PCR conditions were: 93° C. for 2 min 30; 93° C. for 1 min, 60° C. for 1 min, 72° C. for 1 min, 30 cycles; 72° C. for 6 min. After the PCR reaction, the samples were incubated for 60 min at 37° C. with 10 U EcoRI, and 2 μl of the denatured reaction mixture was applied onto a denaturing 5% polyacrylamidc gel containing 35% formamide and 5.6 M urea. Bands were detected by autoradiography.

Based on the 14 chromosomes tested in the first experiment, the deleted Δccr5 allele appeared rather frequent in the Caucasian population. The accurate frequency was further estimated by testing (FIG. 8a) a large cohort of Caucasian individuals, including unrelated members of the CEPH (Centre d'Etude des Polymorphismes Humains) families, part of the IRIBHN staff, and a bank of anonymous DNA samples from healthy individuals collected by the Genetics Department of the Erasme Hospital in Brussels. From a total of more than 700 healthy individuals, the allele frequencies were found to be 0.908 for the wild-type allele, and 0.092 for the mutant allele (Table I). The genotype frequencies observed in the population were not significantly different from the expected Hardy-Weinberg distribution (CCR5/CCR5: 0.827 vs 0.824; CCR5/Δccr5: 0.162 vs 0.167; Δccr5/Δccr5: 0.011 vs 0.008, p>0.999), suggesting that the null allele has no drastic effect on fitness. Using two informative CEPH families, it was confirmed that—the wild-type CCR5 gene and its Δccr5 variant were allelic, and segregated in a normal mendelian fashion (FIG. 8b). Interestingly, a cohort of 124 DNA samples originating from Central Africa (collected from Zaire, Burkina Fasso, Cameroun, Senegal and Benin) and Japan did not reveal a single Δccr5 mutant allele, suggesting that this allele is either absent or very rare in Asian, African black populations (Table I).

The consequences of the existence of a null allele of CCR5 in the normal Caucasian population were then considered in terms of susceptibility to infection by HIV-1. If, as it is predicted, CCR5 plays a major (not redundant) role in the entry of most primary virus strains into cells, then Δccr5/Δccr5 individuals should be particularly resistant to HIV-1 challenge, both in vitro and in vivo. The frequency of the Δccr5/Δccr5 genotype should therefore be significantly lower in HIV-1 infected patients, and increased in exposed-uninfected individuals. Also, if heterozygotes have a statistical advantage due to the lower number of functional receptors on their white blood cells, or to the possible dominant-negative properties of the mutant allele, the frequency of heterozygotes (and mutant alleles) should be decreased in HIV-infected populations. These hypotheses were tested by genotyping a large number of seropositive Caucasian individuals (n=645) belonging to cohorts originating from various hospitals from Brussels, Liège and Paris (Table I). Indeed, it was found that within this large series, the frequency of the null Δccr5 allele was significantly reduced from 0.092 to 0.053 (p<10⁻⁵). The frequency-of heterozygotes was also reduced from 0.162 to 0.106 (p<0.001) and not a single Δccr5/Δccr5 individual could be found (p<0.01).

Altogether, functional and statistical data suggest that CCR5 is indeed the major co-receptor responsible for natural infection by M-tropic HIV-1 strains. Individuals homozygous for the null Δccr5 allele (about 1% of the Caucasian population) have apparently a strong resistance to infection. It is unclear at this point whether resistance to HIV-1 is absolute or relative, and whether resistance will vary depending on the mode of viral contamination. Larger cohorts of seropositive individuals will have to be tested in order to clarify this point. Heterozygotes have a milder though significant advantage: assuming an equal probability of contact with HIV, it can be inferred from Table I that heterozygotes have a 39% reduction in their likeliness of becoming seropositive, as compared to individuals homozygous for the wild-type CCR5 allele. Both a decrease in functional CCR5 receptor number, and a dominant-negative effect of Δccr5 in vivo, comparable to what is observed in the in vitro experiments (FIG. 7) are possible explanations for this relative protection. The mutant allele, which can be regarded as a natural knock-out in human, is not accompanied by an obvious phenotype in homozygous individuals. Nevertheless, the lack of overt phenotype, taken together with the relative protection that characterizes heterozygous subjects, suggests that pharmacological agents that selectively block the ability of HIV-1 to utilize CCR5 as a cofactor, could be effective in preventing HIV-1 infection, and would be predicted not be associated with major side effects resulting from CCR5 inactivation. These pharmaceutical agents could be used with other compounds which are able to block other chemokine receptors used as co-receptors by some HIV-primary isolates in order to infect other cells [47]. The prevalence of the null allele in the Caucasian population raises the question of whether pandemia of HIV (or related viruses using the same co-receptor) have contributed during mankind's evolution to stabilize by selection the mutant ccr5 allele at such a high frequency.

Production of Antibodies Anti-CCR5

Antibodies were produced by genetic immunisation. Six week old females balb/c mice were used. DNA coding for the human CCR5 receptor was inserted in the expression vector pcdna3 under the control of the CMV promotor and 100 μg DNA was injected in the anterior tibial muscle, five days after pre-treatment of this muscle with cardiotoxine (from venom of Naja Nigricolis). Injections were repeated twice at three week intervals. Fifteen days after the last injection, blood was taken from each animal and sera were tested for the presence of anti-CCR5 antibodies.

Test of Sera Using Fluorescence Activated Cell Sorter (FACS)

Sera were tested by fluorescence activated cell sorting using recombinant CHO cells expressing the CCR5 receptor. Briefly, cells were detached using a PBS-EDTA-EGTA solution and incubated into PBS-BSA medium for 30 minutes at room temperature with 5 μl serum on the basis of 100,000 cells per tube. Cells were then washed and incubated for 30 minutes in ice together with anti-mouse antibody labelled with fluorescein. Cells were washed, taken up into 200 μl of a PBS-BSA solution and fluorescence was analysed by FACS (FACSCAN, Becton-Dickinson). 10,000 cells were counted. Wild type CHO or recombinant CHO cells expressing the human CCR2b receptor were used as controls.

When tested by FACS analysis 2 weeks after the last injection (FIG. 9), all the sera from mice immunised with CCR5 CDNA, clearly recognised the native receptor expressed on CHO cells (mean of fluorescence=200), without significant cross reaction with control cells expressing CCR2b (mean of fluorescence=20).

Sera were tested on either a CHO cell line expressing high level of CCR5 receptor (black histogram) or a CHO cell line expressing CCR2b receptor (white histogram) as negative control. Each serum was tested individually.

Antibodies Anti-CCR5 and HIV Infectivity

Peripheral blood mononuclear cells (PBMC) from one donor homozygous from wild type CCR5 gene, were isolated and cultivated 3 days in presence of PHA.

On day 4, 800 μl of cells (10⁵ cells/ml) were incubated with 8 μl of sera from mice immunised with CCR5 CDNA, 30 minutes at 37° C. 1 ml of viral solution (JRCSF HIV strain) is then added and incubated during 2 hours. Cells were then washed twice and cultivated during 15 days.

Aliquot of medium is taken at days 0, 4, 7, 10 and 14 and the dosage of antigen p24 is performed.

14 days after the beginning of the experiment, one serum (serum B0) totally block the production of p24, indicating its ability to block the infection of the lymphocytes by this HIV strain (FIG. 10). Other serums also exhibit a partial or total effect on this infection (serum A2 and B1). All the other sera did not show any effect on this infection.

Production of Monoclonal Antibodies

Mice with the highest title of CCR5 antibodies were selected for monoclonal antibodies production and injected intravenously with 10⁷ recombinant CHO-K1 cells expressing human CCR5 receptors. Three days later, animals were sacrificed and fusion of splenic cells or cells from lymph nodes near the site of injection with SP2/0 myeloma cells, were performed. Fusion protocol used was that of Galfre et al. (Nature 266, 550 (1977)). A selective HAT (hypoxanthine/aminopterin/thymidin) medium is used to select hybridomas and their supernatants are tested by FACS using recombinant CHO cells expressing the human CCR5 receptor, as it was done for the sera. Positives hybridomas are then cloned by limited dilution. Clones that are shown positive by FACS analyses are then expanded and produced in ascites in balb/C mice.

TABLE 1 Seronegative Seropositive Standard Standard Number Frequency error Number Frequency error Chi-squared Genotype: CCR5/CCR5 582 0.827 0.014 645 0.892 0.012 2 degrees of CCR/Δ CCR5 114 0.162 0.014 78 0.108 0.012 freedom CCR/Δ CCR5 8 0.011 0.004 0 0.000 <0.001 17.7 p < 0.0005 Total: 704 1.000 723 1.000 Alleles: CCR5 1278 0.908 0.008 1368 0.946 0.006 1 degree of Δ CCR5 130 0.092 0.008 78 0.054 0.006 freedom p < 0.0005 Total: 1408 1.000 1446 1.000

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                   #             SEQUENCE LISTING (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 17 (2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 792 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:           (A) NAME/KEY: CDS           (B) LOCATION: 240..791     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1: GAATTCCCCC AACAGAGCCA AGCTCTCCAT CTAGTGGACA GGGAAGCTAG CA #GCAAACCT     60 TCCCTTCACT ACAAAACTTC ATTGCTTGGC CAAAAAGAGA GTTAATTCAA TG #TAGACATC    120 TATGTAGGCA ATTAAAAACC TATTGATGTA TAAAACAGTT TGCATTCATG GA #GGGCAACT    180 AAATACATTC TAGGACTTTA TAAAAGATCA CTTTTTATTT ATGCACAGGG TG #GAACAAGG    239 ATG GAT TAT CAA GTG TCA AGT CCA ATC TAT GA #C ATC AAT TAT TAT ACA      287 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr As #p Ile Asn Tyr Tyr Thr   1               5  #                 10  #                 15 TCG GAG CCC TGC CAA AAA ATC AAT GTG AAG CA #A ATC GCA GCC CGC CTC      335 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gl #n Ile Ala Ala Arg Leu              20      #             25      #             30 CTG CCT CCG CTC TAC TCA CTG GTG TTC ATC TT #T GGT TTT GTG GGC AAC      383 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Ph #e Gly Phe Val Gly Asn          35          #         40          #         45 ATG CTG GTC ATC CTC ATC CTG ATA AAC TGC AA #A AGG CTG AAG AGC ATG      431 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Ly #s Arg Leu Lys Ser Met      50              #     55              #     60 ACT GAC ATC TAC CTG CTC AAC CTG GCC ATC TC #T GAC CTG TTT TTC CTT      479 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Se #r Asp Leu Phe Phe Leu  65                  # 70                  # 75                  # 80 CTT ACT GTC CCC TTC TGG GCT CAC TAT GCT GC #C GCC CAG TGG GAC TTT      527 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Al #a Ala Gln Trp Asp Phe                  85  #                 90  #                 95 GGA AAT ACA ATG TGT CAA CTC TTG ACA GGG CT #C TAT TTT ATA GGC TTC      575 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Le #u Tyr Phe Ile Gly Phe             100       #           105       #           110 TTC TCT GGA ATC TTC TTC ATC ATC CTC CTG AC #A ATC GAT AGG TAC CTG      623 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Th #r Ile Asp Arg Tyr Leu         115           #       120           #       125 GCT GTC GTC CAT GCT GTG TTT GCT TTA AAA GC #C AGG ACG GTC ACC TTT      671 Ala Val Val His Ala Val Phe Ala Leu Lys Al #a Arg Thr Val Thr Phe     130               #   135               #   140 GGG GTG GTG ACA AGT GTG ATC ACT TGG GTG GT #G GCT GTG TTT GCG TCT      719 Gly Val Val Thr Ser Val Ile Thr Trp Val Va #l Ala Val Phe Ala Ser 145                 1 #50                 1 #55                 1 #60 CTC CCA GGA ATC ATC TTT ACC AGA TCT CAA AA #A GAA GGT CTT CAT TAC      767 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Ly #s Glu Gly Leu His Tyr                 165   #               170   #               175 ACC TGC AGC TCT CAT TTT CCA TAC A     #                   #              792 Thr Cys Ser Ser His Phe Pro Tyr             180 (2) INFORMATION FOR SEQ ID NO: 2:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1477 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:           (A) NAME/KEY: CDS           (B) LOCATION: 240..1295     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2: GAATTCCCCC AACAGAGCCA AGCTCTCCAT CTAGTGGACA GGGAAGCTAG CA #GCAAACCT     60 TCCCTTCACT ACAAAACTTC ATTGCTTGGC CAAAAAGAGA GTTAATTCAA TG #TAGACATC    120 TATGTAGGCA ATTAAAAACC TATTGATGTA TAAAACAGTT TGCATTCATG GA #GGGCAACT    180 AAATACATTC TAGGACTTTA TAAAAGATCA CTTTTTATTT ATGCACAGGG TG #GAACAAGG    239 ATG GAT TAT CAA GTG TCA AGT CCA ATC TAT GA #C ATC AAT TAT TAT ACA      287 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr As #p Ile Asn Tyr Tyr Thr   1               5  #                 10  #                 15 TCG GAG CCC TGC CAA AAA ATC AAT GTG AAG CA #A ATC GCA GCC CGC CTC      335 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gl #n Ile Ala Ala Arg Leu              20      #             25      #             30 CTG CCT CCG CTC TAC TCA CTG GTG TTC ATC TT #T GGT TTT GTG GGC AAC      383 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Ph #e Gly Phe Val Gly Asn          35          #         40          #         45 ATG CTG GTC ATC CTC ATC CTG ATA AAC TGC AA #A AGG CTG AAG AGC ATG      431 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Ly #s Arg Leu Lys Ser Met      50              #     55              #     60 ACT GAC ATC TAC CTG CTC AAC CTG GCC ATC TC #T GAC CTG TTT TTC CTT      479 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Se #r Asp Leu Phe Phe Leu  65                  # 70                  # 75                  # 80 CTT ACT GTC CCC TTC TGG GCT CAC TAT GCT GC #C GCC CAG TGG GAC TTT      527 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Al #a Ala Gln Trp Asp Phe                  85  #                 90  #                 95 GGA AAT ACA ATG TGT CAA CTC TTG ACA GGG CT #C TAT TTT ATA GGC TTC      575 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Le #u Tyr Phe Ile Gly Phe             100       #           105       #           110 TTC TCT GGA ATC TTC TTC ATC ATC CTC CTG AC #A ATC GAT AGG TAC CTG      623 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Th #r Ile Asp Arg Tyr Leu         115           #       120           #       125 GCT GTC GTC CAT GCT GTG TTT GCT TTA AAA GC #C AGG ACG GTC ACC TTT      671 Ala Val Val His Ala Val Phe Ala Leu Lys Al #a Arg Thr Val Thr Phe     130               #   135               #   140 GGG GTG GTG ACA AGT GTG ATC ACT TGG GTG GT #G GCT GTG TTT GCG TCT      719 Gly Val Val Thr Ser Val Ile Thr Trp Val Va #l Ala Val Phe Ala Ser 145                 1 #50                 1 #55                 1 #60 CTC CCA GGA ATC ATC TTT ACC AGA TCT CAA AA #A GAA GGT CTT CAT TAC      767 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Ly #s Glu Gly Leu His Tyr                 165   #               170   #               175 ACC TGC AGC TCT CAT TTT CCA TAC AGT CAG TA #T CAA TTC TGG AAG AAT      815 Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Ty #r Gln Phe Trp Lys Asn             180       #           185       #           190 TTC CAG ACA TTA AAG ATA GTC ATC TTG GGG CT #G GTC CTG CCG CTG CTT      863 Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Le #u Val Leu Pro Leu Leu         195           #       200           #       205 GTC ATG GTC ATC TGC TAC TCG GGA ATC CTA AA #A ACT CTG CTT CGG TGT      911 Val Met Val Ile Cys Tyr Ser Gly Ile Leu Ly #s Thr Leu Leu Arg Cys     210               #   215               #   220 CGA AAT GAG AAG AAG AGG CAC AGG GCT GTG AG #G CTT ATC TTC ACC ATC      959 Arg Asn Glu Lys Lys Arg His Arg Ala Val Ar #g Leu Ile Phe Thr Ile 225                 2 #30                 2 #35                 2 #40 ATG ATT GTT TAT TTT CTC TTC TGG GCT CCC TA #C AAC ATT GTC CTT CTC     1007 Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Ty #r Asn Ile Val Leu Leu                 245   #               250   #               255 CTG AAC ACC TTC CAG GAA TTC TTT GGC CTG AA #T AAT TGC AGT AGC TCT     1055 Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu As #n Asn Cys Ser Ser Ser             260       #           265       #           270 AAC AGG TTG GAC CAA GCT ATG CAG GTG ACA GA #G ACT CTT GGG ATG ACG     1103 Asn Arg Leu Asp Gln Ala Met Gln Val Thr Gl #u Thr Leu Gly Met Thr         275           #       280           #       285 CAC TGC TGC ATC AAC CCC ATC ATC TAT GCC TT #T GTC GGG GAG AAG TTC     1151 His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Ph #e Val Gly Glu Lys Phe     290               #   295               #   300 AGA AAC TAC CTC TTA GTC TTC TTC CAA AAG CA #C ATT GCC AAA CGC TTC     1199 Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys Hi #s Ile Ala Lys Arg Phe 305                 3 #10                 3 #15                 3 #20 TGC AAA TGC TGT TCT ATT TTC CAG CAA GAG GC #T CCC GAG CGA GCA AGC     1247 Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu Al #a Pro Glu Arg Ala Ser                 325   #               330   #               335 TCA GTT TAC ACC CGA TCC ACT GGG GAG CAG GA #A ATA TCT GTG GGC TTG     1295 Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Gl #u Ile Ser Val Gly Leu             340       #           345       #           350 TGACACGGAC TCAAGTGGGC TGGTGACCCA GTCAGAGTTG TGCACATGGC TT #AGTTTT     1355 TACACAGCCT GGGCTGGGGG TNGGTTGGNN GAGGTCTTTT TTAAAAGGAA GT #TACTGT     1415 TAGAGGGTCT AAGATTCATC CATTTATTTG GCATCTGTTT AAAGTAGATT AG #ATCCGA     1475 TC                   #                   #                   #            1477 (2) INFORMATION FOR SEQ ID NO: 3:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1442 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)     (ix) FEATURE:           (A) NAME/KEY: CDS           (B) LOCATION: 240..884     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3: GAATTCCCCC AACAGAGCCA AGCTCTCCAT CTAGTGGACA GGGAAGCTAG CA #GCAAACCT     60 TCCCTTCACT ACAAAACTTC ATTGCTTGGC CAAAAAGAGA GTTAATTCAA TG #TAGACATC    120 TATGTAGGCA ATTAAAAACC TATTGATGTA TAAAACAGTT TGCATTCATG GA #GGGCAACT    180 AAATACATTC TAGGACTTTA TAAAAGATCA CTTTTTATTT ATGCACAGGG TG #GAACAAGG    239 ATG GAT TAT CAA GTG TCA AGT CCA ATC TAT GA #C ATC AAT TAT TAT ACA      287 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr As #p Ile Asn Tyr Tyr Thr   1               5  #                 10  #                 15 TCG GAG CCC TGC CAA AAA ATC AAT GTG AAG CA #A ATC GCA GCC CGC CTC      335 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gl #n Ile Ala Ala Arg Leu              20      #             25      #             30 CTG CCT CCG CTC TAC TCA CTG GTG TTC ATC TT #T GGT TTT GTG GGC AAC      383 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Ph #e Gly Phe Val Gly Asn          35          #         40          #         45 ATG CTG GTC ATC CTC ATC CTG ATA AAC TGC AA #A AGG CTG AAG AGC ATG      431 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Ly #s Arg Leu Lys Ser Met      50              #     55              #     60 ACT GAC ATC TAC CTG CTC AAC CTG GCC ATC TC #T GAC CTG TTT TTC CTT      479 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Se #r Asp Leu Phe Phe Leu  65                  # 70                  # 75                  # 80 CTT ACT GTC CCC TTC TGG GCT CAC TAT GCT GC #C GCC CAG TGG GAC TTT      527 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Al #a Ala Gln Trp Asp Phe                  85  #                 90  #                 95 GGA AAT ACA ATG TGT CAA CTC TTG ACA GGG CT #C TAT TTT ATA GGC TTC      575 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Le #u Tyr Phe Ile Gly Phe             100       #           105       #           110 TTC TCT GGA ATC TTC TTC ATC ATC CTC CTG AC #A ATC GAT AGG TAC CTG      623 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Th #r Ile Asp Arg Tyr Leu         115           #       120           #       125 GCT GTC GTC CAT GCT GTG TTT GCT TTA AAA GC #C AGG ACG GTC ACC TTT      671 Ala Val Val His Ala Val Phe Ala Leu Lys Al #a Arg Thr Val Thr Phe     130               #   135               #   140 GGG GTG GTG ACA AGT GTG ATC ACT TGG GTG GT #G GCT GTG TTT GCG TCT      719 Gly Val Val Thr Ser Val Ile Thr Trp Val Va #l Ala Val Phe Ala Ser 145                 1 #50                 1 #55                 1 #60 CTC CCA GGA ATC ATC TTT ACC AGA TCT CAA AA #A GAA GGT CTT CAT TAC      767 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Ly #s Glu Gly Leu His Tyr                 165   #               170   #               175 ACC TGC AGC TCT CAT TTT CCA TAC ATT AAA GA #T AGT CAT CTT GGG GCT      815 Thr Cys Ser Ser His Phe Pro Tyr Ile Lys As #p Ser His Leu Gly Ala             180       #           185       #           190 GGT CCT GCC GCT GCT TGT CAT GGT CAT CTG CT #A CTC GGG AAT CCT AAA      863 Gly Pro Ala Ala Ala Cys His Gly His Leu Le #u Leu Gly Asn Pro Lys         195           #       200           #       205 AAC TCT GCT TCG GTG TCG AAA TGAGAAGAAG AGGCACAGG #G CTGTGAGGCT         914 Asn Ser Ala Ser Val Ser Lys     210               #   215 TATCTTCACC ATCATGATTG TTTATTTTCT CTTCTGGGCT CCCTACAACA TT #GTCCTTC     974 CCTGAACACC TTCCAGGAAT TCTTTGGCCT GAATAATTGC AGTAGCTCTA AC #AGGTTGA    1034 CCAAGCTATG CAGGTGACAG AGACTCTTGG GATGACGCAC TGCTGCATCA AC #CCCATCT    1094 CTATGCCTTT GTCGGGGAGA AGTTCAGAAA CTACCTCTTA GTCTTCTTCC AA #AAGCACT    1154 TGCCAAACGC TTCTGCAAAT GCTGTTCTAT TTTCCAGCAA GAGGCTCCCG AG #CGAGCAG    1214 CTCAGTTTAC ACCCGATCCA CTGGGGAGCA GGAAATATCT GTGGGCTTGT GA #CACGGAT    1274 CAAGTGGGCT GGTGACCCAG TCAGAGTTGT GCACATGGCT TAGTTTTCAT AC #ACAGCCG    1334 GGCTGGGGGT GGTTGGGAGG TCTTTTTTAA AAGGAAGTTA CTGTTATAGA GG #GTCTAAA    1394 TTCATCCATT TATTTGGCAT CTGTTTAAAG TAGATTAGAT CCGAATTC   #              1442 (2) INFORMATION FOR SEQ ID NO: 4:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 184 amino  #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4: Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr As #p Ile Asn Tyr Tyr Thr   1               5  #                 10  #                 15 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gl #n Ile Ala Ala Arg Leu              20      #             25      #             30 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Ph #e Gly Phe Val Gly Asn          35          #         40          #         45 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Ly #s Arg Leu Lys Ser Met      50              #     55              #     60 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Se #r Asp Leu Phe Phe Leu  65                  # 70                  # 75                  # 80 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Al #a Ala Gln Trp Asp Phe                  85  #                 90  #                 95 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Le #u Tyr Phe Ile Gly Phe             100       #           105       #           110 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Th #r Ile Asp Arg Tyr Leu         115           #       120           #       125 Ala Val Val His Ala Val Phe Ala Leu Lys Al #a Arg Thr Val Thr Phe     130               #   135               #   140 Gly Val Val Thr Ser Val Ile Thr Trp Val Va #l Ala Val Phe Ala Ser 145                 1 #50                 1 #55                 1 #60 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Ly #s Glu Gly Leu His Tyr                 165   #               170   #               175 Thr Cys Ser Ser His Phe Pro Tyr             180 (2) INFORMATION FOR SEQ ID NO: 5:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 352 amino  #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5: Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr As #p Ile Asn Tyr Tyr Thr   1               5  #                 10  #                 15 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gl #n Ile Ala Ala Arg Leu              20      #             25      #             30 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Ph #e Gly Phe Val Gly Asn          35          #         40          #         45 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Ly #s Arg Leu Lys Ser Met      50              #     55              #     60 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Se #r Asp Leu Phe Phe Leu  65                  # 70                  # 75                  # 80 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Al #a Ala Gln Trp Asp Phe                  85  #                 90  #                 95 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Le #u Tyr Phe Ile Gly Phe             100       #           105       #           110 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Th #r Ile Asp Arg Tyr Leu         115           #       120           #       125 Ala Val Val His Ala Val Phe Ala Leu Lys Al #a Arg Thr Val Thr Phe     130               #   135               #   140 Gly Val Val Thr Ser Val Ile Thr Trp Val Va #l Ala Val Phe Ala Ser 145                 1 #50                 1 #55                 1 #60 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Ly #s Glu Gly Leu His Tyr                 165   #               170   #               175 Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Ty #r Gln Phe Trp Lys Asn             180       #           185       #           190 Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Le #u Val Leu Pro Leu Leu         195           #       200           #       205 Val Met Val Ile Cys Tyr Ser Gly Ile Leu Ly #s Thr Leu Leu Arg Cys     210               #   215               #   220 Arg Asn Glu Lys Lys Arg His Arg Ala Val Ar #g Leu Ile Phe Thr Ile 225                 2 #30                 2 #35                 2 #40 Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Ty #r Asn Ile Val Leu Leu                 245   #               250   #               255 Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu As #n Asn Cys Ser Ser Ser             260       #           265       #           270 Asn Arg Leu Asp Gln Ala Met Gln Val Thr Gl #u Thr Leu Gly Met Thr         275           #       280           #       285 His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Ph #e Val Gly Glu Lys Phe     290               #   295               #   300 Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys Hi #s Ile Ala Lys Arg Phe 305                 3 #10                 3 #15                 3 #20 Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu Al #a Pro Glu Arg Ala Ser                 325   #               330   #               335 Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Gl #u Ile Ser Val Gly Leu             340       #           345       #           350 (2) INFORMATION FOR SEQ ID NO: 6:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 215 amino  #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6: Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr As #p Ile Asn Tyr Tyr Thr   1               5  #                 10  #                 15 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gl #n Ile Ala Ala Arg Leu              20      #             25      #             30 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Ph #e Gly Phe Val Gly Asn          35          #         40          #         45 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Ly #s Arg Leu Lys Ser Met      50              #     55              #     60 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Se #r Asp Leu Phe Phe Leu  65                  # 70                  # 75                  # 80 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Al #a Ala Gln Trp Asp Phe                  85  #                 90  #                 95 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Le #u Tyr Phe Ile Gly Phe             100       #           105       #           110 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Th #r Ile Asp Arg Tyr Leu         115           #       120           #       125 Ala Val Val His Ala Val Phe Ala Leu Lys Al #a Arg Thr Val Thr Phe     130               #   135               #   140 Gly Val Val Thr Ser Val Ile Thr Trp Val Va #l Ala Val Phe Ala Ser 145                 1 #50                 1 #55                 1 #60 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Ly #s Glu Gly Leu His Tyr                 165   #               170   #               175 Thr Cys Ser Ser His Phe Pro Tyr Ile Lys As #p Ser His Leu Gly Ala             180       #           185       #           190 Gly Pro Ala Ala Ala Cys His Gly His Leu Le #u Leu Gly Asn Pro Lys         195           #       200           #       205 Asn Ser Ala Ser Val Ser Lys     210               #   215 (2) INFORMATION FOR SEQ ID NO: 7:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 360 amino  #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: None     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7: Met Leu Ser Thr Ser Arg Ser Arg Phe Ile Ar #g Asn Thr Asn Glu Ser  1               5   #                10   #                15 Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr As #p Tyr Gly Ala Pro Cys             20       #            25       #            30 His Lys Phe Asp Val Lys Gln Ile Gly Ala Gl #n Leu Leu Pro Pro Leu         35           #        40           #        45 Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gl #y Asn Met Leu Val Val     50               #    55               #    60 Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cy #s Leu Thr Asp Ile Tyr 65                   #70                   #75                   #80 Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Ph #e Ile Ile Thr Leu Pro                 85   #                90   #                95 Leu Trp Ala His Ser Ala Ala Asn Glu Trp Va #l Phe Gly Asn Ala Met             100       #           105       #           110 Cys Lys Leu Phe Thr Gly Leu Tyr His Ile Gl #y Tyr Phe Gly Gly Ile         115           #       120           #       125 Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Ty #r Leu Ala Ile Val His     130               #   135               #   140 Ala Val Phe Ala Leu Lys Ala Arg Thr Val Th #r Phe Gly Val Val Thr 145                 1 #50                 1 #55                 1 #60 Ser Val Ile Thr Trp Leu Val Ala Val Phe Al #a Ser Val Pro Gly Ile                 165   #               170   #               175 Ile Phe Thr Lys Cys Gln Lys Glu Asp Ser Va #l Tyr Val Cys Gly Pro             180       #           185       #           190 Tyr Phe Pro Arg Gly Trp Asn Asn Phe His Th #r Ile Met Arg Asn Ile         195           #       200           #       205 Leu Gly Leu Val Leu Pro Leu Leu Ile Met Va #l Ile Cys Tyr Ser Gly     210               #   215               #   220 Ile Leu Lys Thr Leu Leu Arg Cys Arg Asn Gl #u Lys Lys Arg His Arg 225                 2 #30                 2 #35                 2 #40 Ala Val Arg Val Ile Phe Thr Ile Met Ile Va #l Tyr Phe Leu Phe Trp                 245   #               250   #               255 Thr Pro Tyr Asn Ile Val Ile Leu Leu Asn Th #r Phe Gln Glu Phe Phe             260       #           265       #           270 Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Le #u Asp Gln Ala Ile Gln         275           #       280           #       285 Val Thr Glu Thr Leu Gly Met Thr His Cys Cy #s Ile Asn Pro Ile Ile     290               #   295               #   300 Tyr Ala Phe Val Gly Glu Lys Phe Arg Arg Ty #r Ile Ser Val Phe Phe 305                 3 #10                 3 #15                 3 #20 Arg Lys His Ile Xaa Xaa Xaa Phe Cys Lys Gl #n Cys Pro Val Phe Tyr                 325   #               330   #               335 Arg Glu Thr Val Asp Gly Val Thr Ser Thr As #n Thr Pro Ser Thr Gly             340       #           345       #           350 Glu Gln Glu Val Ser Ala Gly Leu         355           #       360 (2) INFORMATION FOR SEQ ID NO: 8:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 355 amino  #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: None     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #8: Met Thr Thr Ser Ile Asp Thr Val Glu Thr Ph #e Gly Thr Thr Ser Tyr  1               5   #                10   #                15 Tyr Asp Asp Val Gly Leu Leu Cys Glu Lys Al #a Asp Thr Arg Ala Leu             20       #            25       #            30 Met Ala Gln Phe Val Pro Pro Leu Tyr Ser Le #u Val Phe Thr Val Gly         35           #        40           #        45 Leu Ile Gly Asn Val Val Val Val Met Ile Le #u Ile Lys Tyr Arg Arg     50               #    55               #    60 Ile Arg Ile Met Thr Asn Ile Tyr Leu Leu As #n Leu Ala Ile Ser Asp 65                   #70                   #75                   #80 Leu Leu Phe Ile Val Thr Leu Pro Phe Trp Th #r His Tyr Val Arg Gly                 85   #                90   #                95 His Asn Trp Val Phe Gly His Gly Met Cys As #n Leu Ile Ser Gly Phe             100       #           105       #           110 Tyr His Thr Gly Leu Tyr Ser Glu Ile Phe Ph #e Ile Ile Leu Leu Thr         115           #       120           #       125 Ile Asp Arg Tyr Leu Ala Ile Val His Ala Va #l Phe Ala Ile Arg Ala     130               #   135               #   140 Arg Thr Val Thr Phe Gly Val Ile Thr Ser Il #e Val Thr Trp Gly Ile 145                 1 #50                 1 #55                 1 #60 Ala Val Ile Ala Ala Leu Pro Glu Phe Ile Ph #e Tyr Glu Thr Glu Glu                 165   #               170   #               175 Leu Phe Glu Glu Thr Ile Cys Ser Ala Leu Ty #r Pro Glu Asp Thr Val             180       #           185       #           190 Tyr Ser Trp Arg His Phe His Thr Ile Arg Me #t Thr Ile Phe Cys Leu         195           #       200           #       205 Val Leu Pro Leu Leu Val Met Ala Ile Cys Ty #r Thr Gly Ile Ile Lys     210               #   215               #   220 Thr Leu Leu Arg Cys Pro Xaa Xaa Xaa Lys Ty #r Lys Ala Ile Arg Leu 225                 2 #30                 2 #35                 2 #40 Ile Phe Val Ile Met Ala Val Phe Phe Ile Gl #u Trp Thr Pro Tyr Asn                 245   #               250   #               255 Val Ala Ile Leu Ile Ser Ser Tyr Gln Ser Le #u Leu Phe Gly Asn Asn             260       #           265       #           270 Cys Glu Arg Ser Lys His Leu Asp Leu Val Me #t Ile Val Thr Glu Val         275           #       280           #       285 Ile Ala Tyr Ser His Cys Cys Met Asn Glu Va #l Ile Tyr Ala Phe Val     290               #   295               #   300 Gly Glu Arg Phe Arg Lys Tyr Ile Arg His Ph #e Phe His Arg His Leu 305                 3 #10                 3 #15                 3 #20 Leu Met His Leu Gly Arg Tyr Ile Pro Phe Le #u Pro Xaa Xaa Xaa Ile                 325   #               330   #               335 Glu Arg Ile Ser Ser Val Ser Pro Ser Thr Al #a Glu Pro Glu Ile Ser             340       #           345       #           350 Ile Val Phe         355 (2) INFORMATION FOR SEQ ID NO: 9:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 355 amino  #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: None     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #9: Met Glu Thr Pro Asn Thr Thr Glu Asp Tyr As #p Thr Thr Thr Glu Phe  1               5   #                10   #                15 Asp Tyr Gly Asp Ala Thr Pro Cys Gln Lys Va #l Asn Glu Arg Ala Phe             20       #            25       #            30 Gly Ala Gln Leu Leu Pro Pro Leu Tyr Ser Le #u Val Phe Val Ile Gly         35           #        40           #        45 Leu Val Gly Asn Ile Leu Val Val Leu Val Le #u Val Gln Tyr Lys Arg     50               #    55               #    60 Leu Lys Asn Met Thr Ser Ile Tyr Leu Leu As #n Leu Ala Ile Ser Asp 65                   #70                   #75                   #80 Leu Leu Phe Ile Phe Thr Leu Pro Phe Trp Il #e Asp Tyr Lys Leu Lys                 85   #                90   #                95 Asp Asp Trp Val Phe Gly Asp Ala Met Cys Ly #s Ile Ile Ser Gly Phe             100       #           105       #           110 Tyr Tyr Thr Gly Leu Tyr Ser Glu Ile Phe Ph #e Ile Ile Leu Leu Thr         115           #       120           #       125 Ile Asp Arg Tyr Leu Ala Ile Val His Ala Va #l Phe Ala Ile Arg Ala     130               #   135               #   140 Arg Thr Val Thr Phe Gly Val Ile Thr Ser Il #e Ile Ile Trp Ala Ile 145                 1 #50                 1 #55                 1 #60 Ala Ile Ile Ala Ser Met Pro Gly Leu Tyr Ph #e Ser Lys Thr Gln Trp                 165   #               170   #               175 Glu Phe Thr His His Thr Cys Ser Leu His Ph #e Pro His Glu Ser Leu             180       #           185       #           190 Arg Glu Trp Lys Leu Phe Gln Ala Leu Lys Le #u Asn Leu Phe Gly Leu         195           #       200           #       205 Val Leu Pro Leu Leu Val Met Ile Ile Cys Ty #r Ile Gly Ile Ile Lys     210               #   215               #   220 Ile Leu Leu Arg Arg Pro Asn Glu Lys Lys Se #r Lys Ala Val Arg Leu 225                 2 #30                 2 #35                 2 #40 Ile Phe Val Ile Met Ile Ile Phe Phe Leu Ph #e Trp Ile Pro Tyr Asn                 245   #               250   #               255 Leu Thr Ile Ile Ile Ser Val Phe Gln Asp Ph #e Leu Phe Thr His Glu             260       #           265       #           270 Cys Glu Gln Ser Arg His Leu Asp Leu Ala Va #l Gln Val Thr Glu Val         275           #       280           #       285 Ile Ala Tyr Thr His Cys Cys Val Asn Glu Va #l Ile Tyr Ala Phe Val     290               #   295               #   300 Gly Glu Arg Phe Arg Lys Tyr Ile Arg Gln Le #u Glu His Arg Arg Val 305                 3 #10                 3 #15                 3 #20 Ala Val His Leu Val Lys Trp Leu Pro Phe Le #u Ser Val Asp Arg Ile                 325   #               330   #               335 Glu Arg Val Ser Ser Thr Ser Pro Ser Thr Gl #y Glu His Glu Ile Ser             340       #           345       #           350 Ala Gly Phe         355 (2) INFORMATION FOR SEQ ID NO: 10:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 360 amino  #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: None     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #10: Met Asn Pro Thr Asp Ile Ala Asp Thr Thr Le #u Asp Glu Ser Ile Tyr  1               5   #                10   #                15 Ser Asn Tyr Tyr Leu Tyr Glu Ser Ile Pro Ly #s Pro Cys Thr Lys Glu             20       #            25       #            30 Gly Ile Lys Ala Phe Gly Glu Leu Phe Leu Pr #o Pro Leu Tyr Ser Leu         35           #        40           #        45 Val Glu Val Phe Gly Leu Ile Gly Asn Ser Va #l Val Val Leu Val Leu     50               #    55               #    60 Phe Lys Tyr Lys Arg Ile Arg Ser Met Thr As #p Val Tyr Leu Leu Asn 65                   #70                   #75                   #80 Leu Ala Ile Ser Asp Leu Leu Phe Val Phe Se #r Leu Pro Phe Trp Gly                 85   #                90   #                95 Tyr Tyr Ala Ala Asp Gln Trp Val Phe Gly Le #u Gly Ile Cys Lys Met             100       #           105       #           110 Ile Ser Trp Met Tyr Leu Val Gly Phe Tyr Se #r Gly Ile Phe Phe Val         115           #       120           #       125 Met Ile Met Ser Ile Asp Arg Tyr Leu Ala Il #e Val His Ala Val Glu     130               #   135               #   140 Xaa Xaa Xaa Ala Arg Thr Ile Ile Tyr Gly Va #l Ile Thr Ser Leu Ala 145                 1 #50                 1 #55                 1 #60 Thr Trp Ser Val Ala Val Phe Ala Ser Leu Pr #o Gly Phe Ile Phe Ser                 165   #               170   #               175 Thr Cys Tyr Thr Glu Arg Asn His Thr Tyr Cy #s Lys Thr Lys Tyr Ser             180       #           185       #           190 Leu Asn Ser Thr Thr Trp Lys Val Leu Ser Se #r Leu Glu Ile Asn Ile         195           #       200           #       205 Leu Gly Leu Val Ile Pro Leu Gly Ile Met Le #u Phe Cys Tyr Ser Met     210               #   215               #   220 Ile Ile Arg Thr Leu Gln His Cys Lys Asn Gl #u Lys Lys Asn Lys Ala 225                 2 #30                 2 #35                 2 #40 Val Lys Met Ile Phe Ala Val Val Val Leu Ph #e Leu Gly Phe Trp Thr                 245   #               250   #               255 Pro Tyr Asn Ile Val Leu Phe Leu Glu Thr Le #u Val Glu Leu Glu Val             260       #           265       #           270 Ile Gln Asp Cys Thr Phe Glu Arg Tyr Leu As #p Tyr Ala Ile Gln Ala         275           #       280           #       285 Thr Glu Thr Leu Ala Phe Val His Cys Cys Le #u Asn Pro Ile Ile Tyr     290               #   295               #   300 Phe Phe Leu Gly Glu Lys Phe Arg Lys Tyr Il #e Ile Gln Leu Phe Lys 305                 3 #10                 3 #15                 3 #20 Xaa Xaa Xaa Gly Leu Phe Val Ile Cys Gln Ty #r Cys Gly Leu Leu Gln                 325   #               330   #               335 Ile Tyr Ser Ala Asp Thr Pro Ser Ser Ser Ty #r Thr Gln Ser Thr Met             340       #           345       #           350 Asp His Asp Leu His Asp Ala Leu         355           #       360 (2) INFORMATION FOR SEQ ID NO: 11:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 54 amino  #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #11: Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Ty #r Gln Phe Trp Lys Asn  1               5   #                10   #                15 Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Le #u Val Leu Pro Leu Leu             20       #            25       #            30 Val Met Val Ile Cys Tyr Ser Gly Ile Leu Ly #s Thr Leu Leu Arg Cys         35           #        40           #        45 Arg Asn Glu Lys Lys Arg     50 (2) INFORMATION FOR SEQ ID NO: 12:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 147 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #12: TTTCCATACA GTCAGTATCA ATTCTGGAAG AATTTCCAGA CATTAAAGAT AG #TCATCTT      60 GGGCTGGTCC TGCCGCTGCT TGTCATGGTC ATCTGCTACT CGGGAATCCT AA #AAACTCG     120 CTTCGGTGTC GAAATGAGAA GAAGAGG           #                   #            147 (2) INFORMATION FOR SEQ ID NO: 13:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 34 amino  #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #13: Phe Pro Tyr Ile Lys Asp Ser His Leu Gly Al #a Gly Pro Ala Ala Ala  1               5   #                10   #                15 Cys His Gly His Leu Leu Leu Gly Asn Pro Ly #s Asn Ser Ala Ser Val             20       #            25       #            30 Ser Lys (2) INFORMATION FOR SEQ ID NO: 14:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #14: TCGAGGATCC AAGATGGATT ATCAAGT           #                   #             27 (2) INFORMATION FOR SEQ ID NO: 15:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #15: CTGATCTAGA GCCATGTGCA CAACTCT           #                   #             27 (2) INFORMATION FOR SEQ ID NO: 16:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #16: CCTGGCTGTC GTCCATGCTG             #                   #                   #20 (2) INFORMATION FOR SEQ ID NO: 17:      (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 27 base  #pairs           (B) TYPE: nucleic acid           (C) STRANDEDNESS: single           (D) TOPOLOGY: linear     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #17: CTGATCTAGA GCCATGTGCA CAACTCT           #                   #             27 

We claim:
 1. An isolated polynucleotide comprising the sequence of SEQ ID NO: 3, or the complement thereof.
 2. The polynucleotide of claim 1, wherein the polynucleotide comprises the sequence of SEQ ID NO:
 3. 3. A method of producing a polypeptide comprising culturing a host cell which comprises a recombinant expression vector comprising the isolated polynucleotide of claim 2, under conditions sufficient to permit expression of said polynucleotide, wherein the polypeptide encoded by said polynucleotide is produced.
 4. The method of claim 3, further comprising isolating said polypeptide.
 5. A method of producing a polypeptide comprising culturing a host cell which comprises the isolated polynucleotide of claim 2, under conditions sufficient to permit expression of said polynucleotide, wherein said polynucleotide is operably linked to a regulatory sequence that controls expression of said polynucleotide.
 6. The method of claim 5, further comprising isolating said polypeptide.
 7. A recombinant expression vector comprising the isolated polynucleotide of claim
 2. 8. A host cell comprising the vector of claim
 7. 9. The host cell of claim 8, wherein said cell is selected from the group consisting of a CHO-K1, HEK293, BHK21, and COS-7 cell.
 10. A genetically engineered host cell comprising the polynucleotide of claim 2, wherein said polynucleotide is operably linked to a regulatory sequence that controls expression of said polynucleotide.
 11. An isolated polynucleotide consisting of the sequence of SEQ ID NO: 1 or 3, or the complement thereof.
 12. A composition comprising the polynucleotide of claim 1 or 11, and a carrier.
 13. A genetically engineered host cell comprising the polynucleotide of claim 11, wherein said polynucleotide is operably linked to a regulatory sequence that controls expression of said polynucleotide.
 14. The polynucleotide of claim 11, wherein the polynucleotide consists of the sequence of SEQ ID NO: 1 or
 3. 15. A method of producing a polypeptide comprising culturing a host cell which comprises a recombinant expression vector comprising the isolated polynucleotide of claim 14, under conditions sufficient to permit expression of said polynucleotide, wherein the polypeptide encoded by said polynucleotide is produced.
 16. The method of claim 15, further comprising isolating said polypeptide.
 17. A method of producing a polypeptide comprising culturing a host cell which comprises the isolated polynucleotide of claim 14, under conditions sufficient to permit expression of said polynucleotide, wherein said polynucleotide is operably linked to a regulatory sequence that controls expression of said polynucleotide.
 18. The method of claim 17, further comprising isolating said polypeptide.
 19. A recombinant expression vector comprising the isolated polynucleotide of claim
 11. 20. A host cell comprising the vector of claim
 19. 21. The host cell of claim 20, wherein said cell is selected from the group consisting of a CHO-K1, HEK293, BHK21, and COS-7 cell. 